下調(diào)miR-10a表達(dá)對(duì)人胰腺癌細(xì)胞AsPC-1遷移和侵襲的影響

張恒 彭輝勇 滿昌峰 徐娟 祁衛(wèi)東 蔣鵬程 范鈺

·論著·

下調(diào)miR-10a表達(dá)對(duì)人胰腺癌細(xì)胞AsPC-1遷移和侵襲的影響

張恒 彭輝勇 滿昌峰 徐娟 祁衛(wèi)東 蔣鵬程 范鈺

目的探討miR-10a表達(dá)對(duì)人胰腺癌AsPC-1細(xì)胞遷移和侵襲力的影響及其作用機(jī)制。方法構(gòu)建針對(duì)miR-10a的小干擾RNA(miR-10a-siRNA)，轉(zhuǎn)染處理人胰腺癌AsPC-1細(xì)胞，同時(shí)設(shè)陰性對(duì)照siRNA(NC-siRNA)組和空白對(duì)照組。采用熒光實(shí)時(shí)定量PCR法檢測(cè)3組細(xì)胞中miR-10a的表達(dá)水平，劃痕實(shí)驗(yàn)檢測(cè)細(xì)胞遷移，Transwell小室檢測(cè)細(xì)胞侵襲能力，ELISA法檢測(cè)各組癌細(xì)胞培養(yǎng)上清液中基質(zhì)金屬蛋白質(zhì)酶13(MMP-13)含量。結(jié)果對(duì)照組、NC-siRNA組和miR-10a-siRNA組AsPC-1細(xì)胞miR-10a表達(dá)水平分別為1.05±0.08、1.03±0.06、0.02±0.01，穿膜細(xì)胞數(shù)分別為(150±2.6)、(145±2.2)、(62±1.8)個(gè)，培養(yǎng)上清中MMP-13表達(dá)量分別為(108.5±2.8)、(107.8±2.5)、(35.8±1.5)pg/ml，miR-10a-siRNA組均顯著低于對(duì)照組和NC-siRNA組，差異均有統(tǒng)計(jì)學(xué)意義(P值均<0.01)。 對(duì)照組、NC-siRNA組和miR-10a-siRNA組AsPC-1細(xì)胞的遷移后間距分別為(385±15)、(395±13) 、(736±18) μm，miR-10a-siRNA組顯著大于對(duì)照組和NC-siRNA組，差異有統(tǒng)計(jì)學(xué)意義(P值均<0.01)。結(jié)論下調(diào)miR-10a表達(dá)可抑制胰腺癌AsPC-1細(xì)胞遷移和侵襲能力，其機(jī)制可能與MMP-13表達(dá)下調(diào)有關(guān)。

胰腺腫瘤； 微RNA； 細(xì)胞運(yùn)動(dòng)； 腫瘤浸潤(rùn)； 基質(zhì)金屬蛋白質(zhì)酶-13； miR-10a

MicroRNAs(miRNAs)的異常表達(dá)與多種人類癌癥的發(fā)生密切相關(guān)[1-8]。近年的研究發(fā)現(xiàn)，miR-10a在人胃癌、甲狀腺癌中高表達(dá)[9]。Ohuchida等[10]報(bào)道，miR-10a在胰腺癌中高表達(dá)，并通過(guò)抑制HOXA1基因的表達(dá)參與細(xì)胞的浸潤(rùn)。本研究采用小干擾RNA(small interfering RNA,siRNA)沉默人胰腺癌AsPC-1細(xì)胞miR-10a的表達(dá)，觀察其對(duì)細(xì)胞遷移、侵襲的影響，探討其機(jī)制。

材料與方法

一、細(xì)胞培養(yǎng)及siRNA轉(zhuǎn)染

人胰腺癌AsPC-1細(xì)胞系購(gòu)自南京凱基生物科技發(fā)展有限公司，常規(guī)培養(yǎng)、傳代。靶向miR-10a的siRNA(miR-10a-siRNA)及陰性對(duì)照siRNA(NC-siRNA)購(gòu)自廣州銳博公司。轉(zhuǎn)染前一天接種適量細(xì)胞至細(xì)胞培養(yǎng)板中，每孔加入不含抗生素的培養(yǎng)基。細(xì)胞密度達(dá)到30%～50%時(shí)用50 μl不含血清的培養(yǎng)基Opti-MEM分別稀釋1.25 μl 20 μmol/L的siRNA貯存液及1 μl脂質(zhì)體，輕輕混勻，室溫孵育5 min后將稀釋的siRNA和脂質(zhì)體輕輕混勻，室溫孵育20 min，加入到細(xì)胞的培養(yǎng)液中輕輕混勻。培養(yǎng)4～6 h，更換新鮮培養(yǎng)基，置37℃的CO2培養(yǎng)箱中繼續(xù)培養(yǎng)48 h。以未轉(zhuǎn)染的AsPC-1細(xì)胞作為對(duì)照。

二、轉(zhuǎn)染效果鑒定

采用Trizol方法抽提兩組轉(zhuǎn)染細(xì)胞及對(duì)照組細(xì)胞總RNA，先逆轉(zhuǎn)錄為cDNA，再采用熒光實(shí)時(shí)定量PCR方法檢測(cè)細(xì)胞miR-10a表達(dá)。參照文獻(xiàn)[11]方法設(shè)計(jì)miR-10a引物、PCR 反應(yīng)條件及計(jì)算miR-10a表達(dá)量。

三、細(xì)胞遷移實(shí)驗(yàn)

先用記號(hào)筆在6孔板背后用直尺均勻地劃?rùn)M線，大約每隔0.5～1 cm一道，橫穿過(guò)孔。取各組對(duì)數(shù)生長(zhǎng)期細(xì)胞，調(diào)整細(xì)胞密度為8×105個(gè)/ml。每孔加入1 ml細(xì)胞懸液，常規(guī)培養(yǎng)至細(xì)胞鋪滿單層。用100 μl槍頭沿培養(yǎng)板底部呈“一”字形劃痕。棄去培養(yǎng)液，用PBS洗細(xì)胞3次以去除劃下的細(xì)胞后繼續(xù)培養(yǎng)24 h，拍照，計(jì)算細(xì)胞未爬入的間距。

四、細(xì)胞侵襲實(shí)驗(yàn)

參照文獻(xiàn)[12]，應(yīng)用Transwell小室檢測(cè)細(xì)胞侵襲力。最后在倒置顯微鏡(×100)下觀察穿膜的細(xì)胞數(shù)。每張膜中央部分和周?chē)糠指麟S機(jī)取3個(gè)視野，計(jì)數(shù)每個(gè)視野內(nèi)的穿膜細(xì)胞數(shù)，取均值。

五、細(xì)胞基質(zhì)金屬蛋白酶13(MMP-13)表達(dá)量測(cè)定

收集各組細(xì)胞培養(yǎng)上清液，采用MMP-13 ELISA試劑盒測(cè)定MMP-13含量，按說(shuō)明書(shū)操作。

六、統(tǒng)計(jì)學(xué)處理

結(jié) 果

一、細(xì)胞miR-10a表達(dá)水平的變化

對(duì)照組、NC-siRNA組和miR-10a-siRNA組AsPC-1細(xì)胞miR-10a表達(dá)量分別為1.05±0.08、1.03±0.06、0.02±0.01(圖1)，miR-10a-siRNA組顯著低于對(duì)照組和NC-siRNA組，差異有統(tǒng)計(jì)學(xué)意義(F=983.280，P=0.000)。

圖1 實(shí)時(shí)PCR的擴(kuò)增曲線(a)和溶解曲線(b)

二、細(xì)胞遷移力的變化

對(duì)照組、NC-siRNA組和miR-10a-siRNA組AsPC-1細(xì)胞遷移后的間距分別為(385±15)、(395±13)、(736±18) μm(圖2)。miR-10a-siRNA組細(xì)胞遷移后的間距顯著大于對(duì)照組和NC-siRNA組，差異有統(tǒng)計(jì)學(xué)意義(F=883.118，P=0.000)。

圖2對(duì)照組(a)、NC-siRNA組(b)、miR-10a-siRNA組(c)AsPC-1細(xì)胞的遷移間距

三、細(xì)胞侵襲力的變化

對(duì)照組、NC-siRNA組和miR-10a-siRNA組AsPC-1細(xì)胞的穿膜細(xì)胞數(shù)分別為(150±2.6)、(145±2.2)、(62±1.8)個(gè)(圖3)，miR-10a-siRNA組明顯少于對(duì)照組和NC-siRNA組，差異有統(tǒng)計(jì)學(xué)意義(F=523.500，P=0.000)。

圖3對(duì)照組(a)、NC-siRNA組(b)、miR-10a-siRNA組(c)的穿膜細(xì)胞

四、細(xì)胞MMP-13的表達(dá)量

對(duì)照組、NC-siRNA組和miR-10a-siRNA組AsPC-1細(xì)胞的培養(yǎng)上清中MMP-13表達(dá)量分別為(108.5±2.8)、(107.8±2.5)、(35.8±1.5)pg/ml，miR-10a-siRNA組顯著低于對(duì)照組和NC-siRNA組(F=463.853，P=0.000)。

討 論

大量證據(jù)表明，miRNAs的異常表達(dá)與多種人類癌癥的發(fā)生密切相關(guān)[1-8]。MiRNAs的發(fā)現(xiàn)和應(yīng)用為胰腺癌等惡性腫瘤的診斷和治療提供了新的靶點(diǎn)。

胰腺癌細(xì)胞轉(zhuǎn)移是一個(gè)復(fù)雜的過(guò)程，遷移和侵襲是重要步驟，也是胰腺癌治療失敗的主要原因之一。積極尋找與胰腺癌侵襲有關(guān)的分子機(jī)制無(wú)疑會(huì)有助于胰腺癌的綜合診療水平。實(shí)驗(yàn)研究表明，在許多實(shí)體瘤如胃癌、甲狀腺癌等瘤組織或細(xì)胞中均高表達(dá)miRNAs[10]，但目前尚不清楚miR-10a在胰腺癌細(xì)胞侵襲中的可能作用。

本研究結(jié)果顯示，與對(duì)照組和NC-siRNA組比較，miR-10a組AsPC-1細(xì)胞的遷移和侵襲能力均顯著減弱，表明miR-10a參與細(xì)胞的遷移及侵襲。

腫瘤的浸潤(rùn)與轉(zhuǎn)移的關(guān)鍵是細(xì)胞外基質(zhì)(extracellular matrix, ECM)成分的降解。MMPs能降解ECM。按照底物特異性，人類MMP主要分為明膠酶、間質(zhì)膠原酶、基質(zhì)溶解素、機(jī)制溶解因子、模型機(jī)制金屬蛋白質(zhì)酶及其他等6類。MMP主要功能是可降解一種或多種ECM成分，調(diào)控血管形成，又可通過(guò)與整合素的相互活化而加強(qiáng)細(xì)胞間的粘附作用。MMP-13是MMPs重要成員之一，它在人許多惡性腫瘤包括人胰腺腺癌細(xì)胞中高表達(dá)[12-15]，且與人胰腺癌侵襲有關(guān)[16]。本研究結(jié)果顯示， miR-10a組細(xì)胞MMP-13表達(dá)量顯著低于對(duì)照組和NC-siRNA組，提示下調(diào)miR-10a表達(dá)后可能通過(guò)抑制MMP-13的表達(dá)而抑制AsPCA-1細(xì)胞的遷移和侵襲能力。

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EffectsofmiR-10adown-regulatedbysiRNAonmigrationandinvasionofhumanpancreaticcancercellAsPC-1

ZHANGHeng,PENGHui-yong,MANChang-feng,XUJuan,QIWei-dong,JIANGPeng-cheng,FANYu.

CancerInstitute,People′sHospitalofZhenjiang,JiangsuUniversity,Zhenjiang212002,China

FANYu,Email：yuf36@sina.com

ObjectiveTo investigate the effects of miR-10a expression on migration and invasion of human pancreatic cancer cells AsPC-1.MethodsSmall interfering RNA targeting at miR-10a(miR-10a-siRNA) was constructed, then it was transfected into pancreatic cancer AsPC-1 cells, and nonsense siRNA (Nc-siRNA) group and blank control group was established. Real time PCR assay was used to detect the expression of miR-10a in the 3 groups, and wound healing assay and Transwell assay were used to determine the migration and invasion abilities of cancer cells. The amount of matrix metalloproteinase-13 (MMP-13) in supernatant of cancer cell culture of each group was examined by ELISA assay.ResultsThe miR-10a levels in control group, NC-siRNA group and miR-10a-siRNA group were 1.05±0.08, 1.03±0.06, 0.02±0.01; and the number of transmembrane cell were (150±2.6), (145±2.2), (62±1.8), the levels of MMP-13 in the supernatant were (108.5±2.8), (107.8±2.5), (35.8±1.5)pg/ml. The values were significantly lower in miR-10a-siRNA group than those in control group and NC-siRNA group (P<0.01). The distance of cultured clone in miR-10a treated cancer cells (736±18 μm) was significantly longer than those in the controls (385±5 μm) and NC-siRNA group (395±13 μm,P<0.01).ConclusionsDown-regulation of miR-10a by siRNA may inhibit migration and invasion of pancreatic cancer AsPC-1 cells, and the down-regulated expression of MMP-13 may be one of the important mechanisms.

Pancreatic neoplasms; MicroRNAs; Cell movement; Neoplasm invasiveness; Matrix metalloproteinase-13; miR-10a

2013-09-29)

(本文編輯：呂芳萍)

10.3760/cma.j.issn.1674-1935.2013.06.004

江蘇省普通高校研究生科研創(chuàng)新計(jì)劃項(xiàng)目(CXLX12_0679)；江蘇大學(xué)臨床科技基金(JLY2010007)

212002 鎮(zhèn)江，江蘇大學(xué)附屬人民醫(yī)院腫瘤研究所

范鈺，Email：yuf36@sina.com